Biosynthetic pathway of penicillin
The first step in the biosynthesis of penicillin involves the formation of the tripeptide .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine (ACV) from L-.alpha.-aminoadipic acid, L-cysteine and L-valine (Fawcett et al., Biochem. J., 157, p. 651-660, 1976). The reaction is catalyzed by a multifunctional enzyme .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase) with ATP and Mg.sup.2+ as co-factors (Banko et al., J. Am. Chem. Soc., 109, p. 2858-2860, 1987).
ACV synthetase (ACVS) has been purified from Aspergillus nidulans (Van Liempt et al., J. Biol. Chem., 264, p. 3680-3684, 1989), Cephalosporium acremonium (Baldwin et al., J. Antibiot., 43, p. 1055-1057, 1990) and Streptomyces clavuligerus (Jensen et al., J. Bacteriol., 172, p. 7269-7271, 1990, and Zhang et al., Biotechnol. Lett., 12, p. 649-654, 1990). The purification of ACV synthetase from Penicillium chrysogenum has not been published. However, ACV synthetase from P. chrysogenum has been cloned by Diez et al. (J. Biol. Chem., 265, p. 16358-16365, 1990).
The linear tripeptide, ACV, is converted to isopenicillin N (IPN) in the presence of isopenicillin N synthase (also referred to as cyclase or isopenicillin N synthetase (IPNS)), ferrous ions, oxygen and an electron donor (lag ascorbate). Isopenicillin N synthase was first isolated from P. chrysogenum by Ramos et al. (Antimicrobial Agents and Chemotherapy, 27, p. 380-387, 1985) and the isopenicillin N synthase structural gene from P. chrysogenum cloned by Carr et al. (Gene, 48, p. 257-266, 1986).
These first two steps in the biosynthesis of penicillins are common in penicillin and cephalosporin producing fungi and bacteria.
In some fungi, for example in P. chrysogenum and in A. nidulans, the .alpha.-aminoadipyl side chain of isopenicillin N can be replaced by other side chains of intracellular origin or exogenously supplied. The exchange is catalyzed by an acyltransferase (referred to as acyl-coenzyme A: isopenicillin N acyltransferase or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase). It is still unclear whether this conversion proceeds in vivo by a two-step reaction in which first the L-.alpha.-aminoadipyl side chain is removed to yield 6-aminopenicillanic acid (6-APA) followed by the acylation step, or the conversion is a direct exchange of the side chains. Purified acyltransferase from P. chrysogenum has both an isopenicillin N-amidohydrolase activity and an acyl-coenzyme A:6-aminopenicillanic acid acyltransferase activity Alvarez et al. Antimicrobial Agents and Chemotherapy, 31, p. 1675-1682, 1987).
The genes coding for ACV synthetase (pcbAB), isopenicillin N synthase (pcbC) and acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (penDE) are found in the same cluster in P. chrysogenum and A. nidulans (Diez et al., J. Biol. Chem., 265, p. 16358-16365, 1990, and Smith et al., Bio/Technology, a, p. 39-41, 1990).
Amplification of the pcbC-penDE gene cluster of P. chrysogenum Wis 54-1255, coding for isopenicillin N synthase (IPNS) and acyltransferase (AT), respectively, led to as much as a 40% improvement in production yields (Veenstra et al., J. Biotechnol., 17, p. 81-90, 1991). Increased antibiotic yields were also reported in A. nidulans transformants containing multiple copies of pcbAB (coding for ACV synthetase (ACVS)) and pcbC genes (coding for isopenicillin N synthetase (INPS)) (McCabe et al., J. Biotechnol., 17, p. 91-97, 1991).
EP 200425 (Eli Lilly) discloses vectors encoding isopenicillin N synthetase (IPNS). The vectors permit high level expression of IPNS in C. acremonium and E. coli. According to the disclosure the Cephalosporium vectors are useful for strain improvement, to increase efficiency and yield in fermentations for the production of penicillin and cephalosporin antibiotics. The vectors may also be modified to give vectors for increasing the production yields and efficiency of P. chrysogenum, Streptomyces clavuligerus etc. in fermentations.
EP 357119 (Gist Brocades) discloses the clustered antibiotic biosynthetic genes encoding IPNS, AT and ACVS and are advantageously employed for improvement of production of the antibiotic in microorganisms and for the isolation of other genes involved in the biosynthesis of the antibiotic. The invention is exemplified with improved production of penicillin in P. chrysogenum, with the isolation of another clustered biosynthetic gene(s) and with the expression of clustered penicillin biosynthetic genes in Acremonium chrysogenum.
Activation of side chain
In order to replace the .alpha.-aminoadipic acid side chain in the acyltransferase catalyzed reaction, the carboxylic acid group of the new side chain has to be activated. This activation is one of the least well understood parts of the biosynthesis of penicillins. Two theories have been proposed.
The most widely accepted theory is that the enzyme catalyses the esterification of carboxylic acids into coenzyme A thioesters by a two-step mechanism that proceeds through the pyrophosphorolysis of ATP (adenosine triphosphate), in the presence of Mg.sup.2+. Firstly, the carboxylic acid (the new side chain), ATP and the enzyme forms a complex, leading to an acyl-AMP-enzyme complex. Secondly, this complex reacts with coenzyme A to liberate acyl-coenzyme A and AMP (adenosine monophosphate).
The other theory is based on the formation of an acyl-S-glutathione intermediate, which may be transformed to the corresponding acyl-coenzyme A ester (Ferrero et al., J. Antibiot., 43, p. 684-91, 1990.
A phenacyl:coenzyme A ligase from P. chrysogenum able to catalyze the synthesis of phenoxyacetyl-coenzyme A and phenylacetyl-coenzyme A in the presence of ATP, Mg.sup.2+, coenzyme A and phenoxy-acetic acid or phenylacetic acid has been described by Brunner, Rohr and Zinner (Hoppe-Seyler's Z. Physiol. Chem., 349, p. 95-103, 1968), Brunner and Rohr (Methods Enzymol., 43, p. 476-481, 1975; Kogekar and Deshpande, Ind. J. Biochem. Biophys., 19, p. 257-261, 1982, and by Kurzatkowski, Med. Dosw. Mikrobiol., 33, p. 15-29, 1981). According to Brunner et al., the ligase shows similar degrees of activity towards phenylacetic acid, phenoxyacetic acid and acetic acid. However, the enzyme was never purified to homogeneity.
Martinez-Blanco et al. (J. Biol. Chem., 267, p. 5474-5481, 1992) have described an acetyl-coenzyme A synthetase from P. chrysogenum Wis 54-1255 which not only accepts acetic acid but also phenylacetic acid as substrates in the synthesis of the corresponding acyl-coenzyme A esters just like the ligase described by Brunner et al. However, the activity towards phenoxyacetic acid is not described by Martinez-Blanco et al. According to Martinez-Blanco et al., the acetyl-coenzyme A synthetase is a homo-dimer (.alpha..sub.2) having a molecular weight of 139,000 Dalton as determined by gel filtration and of 70,000 Dalton as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and an isoelectric point: between pH 5.6 and 6.0.
The gene coding for the acetyl-coenzyme A synthetase of Martinez-Blanco et al. has been characterized by Martinez-Blanco et al. (Gene, 130, p. 265-270, 1993). The gene which was designated acuA contains five introns and codes for a polypeptide of 669 amino acids. This polypeptide has a molecular weight of 74,287.
Gouka et al. (Appl. Microbiol. Biotechnol., 38, p. 514-519, 1993) and Van Hartingsveldt et al. (WO 92/07079) have described the isolation and sequence of an acetyl-coenzyme A synthetase (facA) gene from P. chrysogenum coding for a protein of 669 amino acids corresponding to a molecular weight of approximately 74,000 Dalton (using an average molecular weight of 110 g/mol for each amino acid). The gene sequences of the facA gene of Gouka et al. and of acuA of Martinez-Blanco et al. showed no differences.
A detailed characterization of the phenylacyl-coenzyme A ligases described by Brunner et al. and by Kogekar and Deshpande has not been published.
In its activated form, the new side chain which is to replace the .alpha.-aminoadipic acid side chain in the acyl transferase catalyzed reaction may be in the form of a coenzyme A thioester or another thioester, since other thioesters (e.g. acyl-S-cysteinyl-glycine and acyl-S-glutathione) have been reported to be substrates for the acyltransferase. As a further possibility, the dipeptide (cysteinyl-glycine) may be substituted by CoASH in a non-enzymatic reaction before it enters into the acyltransferase reaction (Ferrero et al., J. Biol. Chem., 265, p. 7084-7090, 1990).
Once it is formed, the thionylgroup of the thioesters may exchange rapidly with other thiols (e.g. mercaptoethanol, 1,4-dithithreitol, ACV and coenzyme A) in a non-enzymatic reaction.
Ligases in general
Ligases, belonging to the enzyme subclass 6.2.1., Acid-Thiol ligases (enzyme Nomenclature, Academic Press, inc., 1992) also referred to as acyl-coenzyme A synthetases or acyl-coenzyme A thiokinases, catalyze the formation of acyl-coenzyme A thioesters from a carboxylic acid and coenzyme A in the presence of ATP and Mg.sup.2+.
Several ligases or acyl-coenzyme A synthetases from various sources have been identified e.g. acetyl-coenzyme A synthetase, propionyl-coenzyme A synthetase (Groot, Biochim. Biophys. Acta, 441, p. 260-267, 1976); butyryl-coenzyme A synthetase (Wanders et al., J. Biol. Chem., 240, p. 29-33, 1965); medium-chain, long-chain and very long-chain fatty acyl-coenzyme A synthetases (Waku, Biochim. Biophys. Acta, 1124, p. 101-111, 1992, review); benzoyl-coenzyme A ligase from Pseudomonas sp (Auburger, Appl. Microbiol. Biotechnol., 37, p. 789-795, 1992) and phenylacetyl-coenzyme A ligases (Martinez-Blanco et al., J. Biol. Chem., 265, p. 7084-7090, 1990; and Vitovski (FEMS Microbiol. Letters, 108, p. 1-6, 1993). Most of them have broad substrate specificities.
The acyl-coenzyme A synthetases (ligases) are generally key enzymes in the primary metabolism of fatty acids and acetic acid and in the initial steps in the degradation of aromatic acids where they through the formation of the energy-rich thioester bond activate the acyl group of the carboxylic acid.
In vivo production of .beta.-lactams
Many of the so called natural .beta.-lactams (e.g. penicillin DF, isopenicillin N, 6-APA, cephalosporin C etc.) are unstable, difficult to purify from the fermentation broth, have only limited antibiotic effect, and/or are produced in low yield.
Replacing the side chains of the natural .beta.-lactams with e.g. phenoxyacetic acid or phenylacetic acid leads to the formation of penicillins (penicillin V and penicillin G, respectively), which are more stable, easier to isolate and having a higher antibiotic activity.
The only directly fermented penicillins of industrial interest are penicillin V and penicillin G, produced by adding phenoxyacetic acid or phenyllactic acid, respectively to the fermentation tank. Addition of alternative precursors, for example 2-thiopheneacetic acid or 3-thiopheneacetic acid, during the fermentation of P. chrysogenum leads to other penicillins. Some added precursors e.g. 1-phenyl-n-alkanes or 1-phenoxy-n-alkanes may be partly metabolized within the P. chrysogenum cells before being used a substrate for the acyltransferase in the production of penicillins (Szarka, Advances in Biotechnology, 3, p. 167-173, 1980; Szarka et al., U.S. Pat. No. 4,250,258; and Szarka et al., U.S. Pat. No. 4,208,481). In the biosynthesis of the so-called natural penicillins: e.g. penicillin DF, penicillin K, penicillin F and penicillin H; the side chains: hexanoic acid, octanoic acid, 3-hexenoic acid, and heptanoic acid, respectively, are presumably derived from the primary metabolism.
In some cases the phenoxyacetyl or the phenylacetyl side chain also acts as a protection group during the fermentation and recovery of the .beta.-lactam, as the isolated penicillin V or G are hydrolysed by an organic chemical or enzymatic process to form 6-APA, which, in turn is the basic building block for new semi-synthetic penicillins having improved pharmacological properties compared to the natural penicillins as-well as the penicillin V or G.
Likewise in the production of cephalosporins, the natural cephalosporins are of limited pharmaceutical value, as they too are difficult to isolate and have only a low antibiotic effect. Furthermore, they are difficult to transform into new cephalosporins of higher pharmaceutical value.
A number of steps have to be carried out, in order to transform the fermented cephalosporin into the desired antibiotic. e.g. in the production of the oral cephalosporins cephalexin and cefadroxil, the starting point is penicillin V or G, which is then transformed into a cephalosporin by a series of chemical reactions, keeping the phenoxyacetyl or phenylacetyl side chain as a protection group. After the ring expansion V-DCA or G-DCA (V/G-deacetoxycephalosporanic acid), respectively, is formed, the side-chain is removed by hydrolysis in a process similar to the hydrolysis of penicillin V or G. Finally, a new sidechain (e.g. D-phenylglycine or D-p-hydroxy-phenylglycine) is added by an organic chemical process.
In the production of cephalosporins from cephalosporin C, the D-.alpha.-aminoadipic acid may be removed either by chemical hydrolysis or by a two step organic chemical enzymatic process. The resulting 7-ACA (7-amino cephalosporanic acid) is then acylated to form the desired product.
Both the reactions leading to the formation of V-DCA, G-DCA and the hydrolysis of cephalosporin C to 7-ACA are carried out in industrial scale but they are difficult to control, expensive and yields generally are low.
In order to circumvent this problem, several alternatives have been suggested some of which involves the transformation of P. chrysogenum with the epimerase and expandase from e.g. Streptomyces, as described by C. Cantwell et al., 248, p. 283-289, 1992. They demonstrated the transformed P. chrysogenum was able to produce deacetoxycephalosporanic acid. However, no V-DCA was found. Cantwell suggested the expandase may be modified by genetic engineering in order to change its substrate specificity to accept penicillin V or G as substrate. Then the V/G-DCA may be produced directly by fermentation.
S. Gutierrez et al., Mol. Gen. Genet., 225, p. 56-64, 1991, transformed C. acremonium with the acyltransferase from P. chrysogenum, and were able to detect penicillin G formation by the transformed C. acremonium, but the presence of G-DCA was not proved.
EP 532341 (Merck & Co, Inc.) describes the fermentation of adipoyl-7-ADCA (adipoyl-7-aminodesacetoxycephalosporanic acid) in P. chrysogenum transformed with the expandase from Streptomyces clavuligerus. The adipoyl-7-ADCA can then be extracted from the fermentation broth and hydrolysed by an adipoyl acylase from e.g. Pseudomonas.
ES 2,016,476 discloses in vitro bio-chemical preparation of phenylacetyl-coenzyme A and benzyl penicillin (penicillin G). Phenylacetyl-coenzyme A is made by incubating phenyl-acetyl-coenzyme A ligase from Pseudomonas putida with ATP, phenylacetic acid and MgCl.sub.2 at 10-45.degree. C. and pH 5-10 for 10-180 minutes. Benzyl penicillin is made by incubating the phenylacetyl-coenzyme A ligase and acyl-coenzyme A:6-aminopenicillanic acid acyl transferase from Penicillium chrysogenum with 6-aminopenicillanic acid and phenylacetic acid, ATP, coenzyme A and MgCl.sub.2 at 10-40.degree. C. and pH 5.5-9 for 30-180 minutes.
ES 2,033,590 discloses ji vitro production of different penicillins derived from benzyl penicillin (penicillin G). The production comprises incubating an enzyme system containing phenylacetyl-coenzyme A ligase from Pseudomonas putida and acyl-coenzyme A-aminopenicillanic acid acyltransferase of Penicillium chrysogenum, and substrates, such as 6-amino penicillanic acid, ATP, coenzyme A, MgCl.sub.2, dithiotreitol (DTT) and precursors of the penicillins.
In the Prior art processes have been disclosed for the production of certain penicillins and cephalosporins that include in vitro steps. Mostly these processes are difficult to control, cumbersome and/or expensive.